Chrom xp c18 trap column

Peptide‐containing HPLC fractions were dried, resuspended in a solvent composed of 10% acetic acid, 2% ACN, and iRT peptides (Biognosys, Schlieren, Switzerland) as internal standards. Fractions were applied individually to an Eksigent nanoLC 415 nanoscale RP‐HPLC (AB Sciex, Framingham, Massachusetts, USA), including a 5‐mm‐long, 350 μm of internal diameter Chrom XP C18 trap column with 3 μm particles and 120 Å pores, and a 15‐cm‐long ChromXP C18 separation column (75 μm internal diameter) packed with the same medium (AB Sciex, Framingham, Massachusetts, USA). An ACN gradient was run at pH 2.5 using a two‐solvent system. Solvent A was 0.1% formic acid in water, and solvent B was 0.1% formic acid in 95% ACN in water. The column was pre‐equilibrated at 2% solvent B. Samples were loaded at 5 μl/min flow rate onto the trap column and run through the separation column at 300 nl/min with two linear gradients: 10–40% B for 70 min, followed by 40–80% B for 7 min.
The column effluent was ionized using the nanospray III ion source of an AB Sciex TripleTOF 5600 quadrupole time‐of‐flight mass spectrometer (AB Sciex, Framingham, MA, USA) with the source voltage set to 2,400 V. Information‐dependent analysis (IDA) method was used for data acquisition (Kaabinejadian et al, 2022 (link)). PeakView Software version 1.2.0.3 (AB Sciex, Framingham, MA, USA) was used for data visualization.

Approximately 8 μg of tryptic digest were separated on a NanoLC 425 System (Sciex). A flow of 5 μl min⁻¹ was used with trap-elute setting using a ChromXP C18 trap column 0.5 × 10 mm, 5 μm, 120 Å (catalogue number 5028898, Sciex). Tryptic peptides were eluted from a ChromXP C18 column 0.3 × 150 mm, 3 μm, 120 Å (catalogue number 5022436, Sciex) using a 43 min gradient from 4% to 32% B with 1 h total run. Mobile phase solvents consisted of 92.9% water, 2% acetonitrile, 5% dimethyl sulfoxide and 0.1% formic acid (A phase) and 92.9% acetonitrile, 2% water, 5% dimethyl sulfoxide and 0.1% formic acid (B phase). Mass spectrometry analysis was performed using Sequential Window Acquisition of all Theoretical (SWATH) acquisitions on a TripleTOF 6600 System equipped with a DuoSpray Source and 25 mm inner diameter electrode (Sciex). Variable Q1 window SWATH acquisition methods (100 windows) were built-in high-sensitivity tandem mass spectrometry mode with Analyst TF Software (v1.7).